Bone tissue is a composite of proteins, cells and minerals known as bone matrix. In a living animal, cells called osteoblasts build bone matrix and cells called osteoclasts break down and resorb bone matrix. Osteoblasts arise from mesenchymal stem cells and produce bone matrix during development, after bone injury, and during the normal bone remodelling that occurs throughout life. Osteoclasts differentiate from hematopoietic precursors of the monocyte-macrophage lineage and resorb bone matrix to support normal bone remodelling, in response to injury or stress, and in response to various disease states.
The equilibirium between the construction and resorption of bone matrix is regulated by numerous factors. One of the systems that regulates bone physiology is the OPG/RANKL/RANK system. This system includes three factors: osteoprotegerin (“OPG”); receptor activator of NF-κB (“RANK”); and RANK ligand “RANKL”).
RANK ligand, or RANKL, also variously art-known as ODF (osteoclast differentiation factor), OPGL (osteoprotegerin ligand) and TRANCE (TNF-related activation-induced cytokine), and by other designations, is a member of the TNF ligand family. RANKL exists in two forms: a cellular membrane-bound form and a soluble form. RANKL mRNA exhibits its highest level of expression in bone. The major role of RANKL in bone is to stimulate osteoclast differentiation and activity, and to inhibit osteoclast apoptosis. In the presence of low levels of macrophage-colony stimulating factor (M-CSF), RANKL appears to be both necessary and sufficient for the complete differentiation of osteoclast precursor cells into mature osteoclasts. RANKL mRNA is also expressed in lymphoid tissues, such as the lymph node, thymus, spleen, fetal liver and Peyer's patches. In addition, RANKL has a number of effects on immune cells. These effects include the activation of c-Jun N-terminal kinase (JNK) in T cells, inhibition of apoptosis of dendritic cells, induction of cluster formation by dendritic cells and effects on cytokine-activated T cell proliferation.
The RANKL/RANK signaling pathway has been characterized. RANKL, expressed on the surface of pre-osteoblast/stromal cells or in soluble form, binds to RANK, which is expressed on osteoclast precursor cells. This binding promotes the differentiation of osteoclast precursor cells into mature osteoclasts. Macrophage colony stimulating factor (M-CSF), which binds to its receptor, c-Fms, on preosteoclastic cells, appears to be involved in osteoclast development because it is the primary determinant of the pools of these precursor cells. OPG is a soluble receptor for RANKL, and can block the effects of RANKL by acting as a “decoy” binding target. In addition, a number of cytokines, including TNF-α and IL-1, modulate the system, for example, by stimulating M-CSF production or by increasing RANKL expression.
Proper functioning of the OPG/RANKL/RANK system is essential for bone metabolism, immune functions and vascular functions. Disruptions in this system have been implicated in various skeletal and immune disorders, such as rheumatoid arthritis, osteoporosis and osteopetrosis. Antagonists of RANKL can be used to treat osteoporosis and other conditions mediated by such RANKL/RANK interactions. Assays for the identification of such antagonists to human, mouse and rat RANKL have been enabled by the isolation of human, mouse and rat RANKL polypeptides. Because the antigenic (extracellular) domain of RANKL varies among species, species specific RANKL polypeptides are preferred for identifying species specific RANKL antagonists.
The RANKL proteins and encoding genes for several mammalian species are known. For example, human RANKL protein and encoding nucleic acid is described, for example, by U.S. Pat. No. 6,242,213. Rat RANKL protein and encoding nucleic acid is described, for example, by International published patent appl. No. WO 01/23549. Murine RANKL protein and encoding nucleic acid is described, for example, by co-owned U.S. Pat. No. 6,242,586. Methods of modulating the effects of RANKL from several non-canine sources are described, for example, by co-owned U.S. Pat. No. 6,525,180, by U.S. Pat. No. 6,316,408, and by Halkier et al., in published international patent application (WO0015807A1, March 2000), However, there remains a longfelt and heretofore unmet need in the art for the identification and production of canine RANKL, as well as for new methods and antagonists for modulating the effects of canine RANKL in canine and other animal, e.g., mammalian species.
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.